Using Bacillus subtilis as a gram-positive model, we propose to investigate the mechanism of proteolytic control that is applied in response to oxidative stress. Proteolytic control is essential for protein homeostasis and also plays a key role in modulating the levels of specific regulatory factors. One such factor, Spx, is a global transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Its concentration is elevated when cells undergo oxidative stress. When stress is alleviated, Spx is degraded by the ATP-dependent protease, ClpXP, a process that requires the substrate recognition factor, YjbH. Spx interacts directly with YjbH, which promotes ClpXP-catalyzed Spx proteolysis. Our recent studies show that a small peptide YirB binds to YjbH thereby releasing Spx from proteolytic control. One of our goals is to study the interactions between these four components, Spx/YjbH/ClpX/YirB, both in vivo and in vitro during oxidative stress and as stress subsides. This will be determined using active, affinity-tagged versions of the binding partners for pull down interaction experiments to be conducted with in vivo samples and in vitro reactions. In addition, to gaining in-depth understanding of the interactions, I will undertake structural studies of the key player YjbH and its complexes with Spx and YirB. During this award period, I intend to gain skills in bacterial genetics and knowledge to design approaches to elucidate the complexities of regulated proteolysis, while utilizing my advanced expertise in crystallography to define in detail the interactions between the protein components at the molecular level. PUBLIC HEALTH RELEVANCE: Infectious microorganisms must defend themselves from stress brought about by toxic agents such as oxidants produced by the host immune system. Bacteria can regulate their response to oxidants by precisely and rapidly changing the levels of specific factors that govern the processes that prevent and/or alleviate stress. Our application is to characterize the system of control that determines the concentration of an important regulator of the bacterial stress response, the protein called Spx. I will define in detail the structure and function of the factors that control the degradation of Spx before and during recovery from oxidant-induced stress.